Process analytical technology (PAT) has been defined by the Food and Drug Administration (FDA) as a system for designing, analyzing, and controlling manufacturing through timely measurements to ensure final product quality. Based on quality-by-design (QbD) principles, real-time or near-real-time data monitoring is essential for timely control of critical quality attributes (CQAs) to keep the process in a state of control. To facilitate next-generation continuous bioprocessing, deployment of PAT tools for real-time monitoring is integral for process understanding and control. Real-time monitoring and control of CQAs is essential to keep the process within the design space and align with the guiding principles of QbD. The contents of this manuscript are pertinent to the online/at-line monitoring of upstream titer and downstream product quality with timely process control. We demonstrated that a UPLC system interfaced with a process sample manager (UPLC-PSM) can be utilized to measure titer and CQAs directly from bioreactors and downstream unit operations, respectively. We established online titer measurements from fed-batch and perfusion-based alternating tangential flow (ATF) bioreactors as well as product quality assessments of downstream operations for real-time peak collection. This integrated, fully automated system for online data monitoring with feedback control is designed to achieve desired product quality.
Chemical group-transfer reactions by hydrolytic enzymes have considerable importance in biocatalytic synthesis and are exploited broadly in commercial-scale chemical production. Mechanistically, these reactions have in common the involvement of a covalent enzyme intermediate which is formed upon enzyme reaction with the donor substrate and is subsequently intercepted by a suitable acceptor. Here, we studied the glycosylation of glycerol from sucrose by sucrose phosphorylase (SucP) to clarify a peculiar, yet generally important characteristic of this reaction: partitioning between glycosylation of glycerol and hydrolysis depends on the type and the concentration of the donor substrate used (here: sucrose, α-D-glucose 1-phosphate (G1P)). We develop a kinetic framework to analyze the effect and provide evidence that, when G1P is used as donor substrate, hydrolysis occurs not only from the β-glucosyl-enzyme intermediate (E-Glc), but additionally from a noncovalent complex of E-Glc and substrate which unlike E-Glc is unreactive to glycerol. Depending on the relative rates of hydrolysis of free and substrate-bound E-Glc, inhibition (Leuconostoc mesenteroides SucP) or apparent activation (Bifidobacterium adolescentis SucP) is observed at high donor substrate concentration. Using G1P at a concentration excluding the substrate-bound E-Glc, the product ratio changes to a value consistent with reaction exclusively through E-Glc, independent of the donor substrate used. Collectively, these results give explanation for a kinetic behavior of SucP not previously accounted for, provide essential basis for design and optimization of the synthetic reaction, and establish a theoretical framework for the analysis of kinetically analogous group transfer reactions by hydrolytic enzymes.
Affinity precipitation using stimulus-responsive biopolymers such as Elastin-like Polypeptides (ELPs) have been successfully employed for the purification of monoclonal antibodies. In the current work, we extend these studies to the development of an ELP-peptide fusion for the affinity precipitation of the therapeutically relevant small non-mAb biologic, AdP. A 12-mer affinity peptide ligand (P10) was identified by a primary phage biopanning followed by a secondary in-solution fluorescence polarization screen. Peptide P10 and AdP interacted with a KD of 19.5 µM. A fusion of P10 with ELP was then shown to be successful in selectively capturing the biologic from a crude mixture. While pH shifts alone were not sufficient for product elution, the use of pH in concert with fluid phase modifiers such as NaCl, arginine or ethylene glycol was successful. In particular, the use of pH 8.5 and an arginine concentration of 500 mM enabled > 80% product recovery. The overall process performance evaluated by SDS-PAGE and reversed-phase UPLC analyses, indicated the successful single-step purification of the biologic from an E. coli lysate resulting in ~90% purity and >80% recovery. These results demonstrate that phage display can be readily employed to identify a peptide ligand capable of successfully carrying out the purification of a non-antibody biological product using ELP-based affinity precipitation.
Protein lipoylation is essential for the function of many key enzymes, but barely studied kinetically. Here, the two-step reaction cascade of H protein lipoylation catalyzed by the multifunctional enzyme lipoate-protein ligase A (LplA) was quantitatively and differentially studied. We discovered new phenomena and unusual kinetics of the cascade: (1) the speed of the first reaction is faster than the second one by two orders of magnitude, leading to high accumulation of the intermediate Lip-AMP; (2) Lip-AMP is hydrolyzed, but only significantly at the presence of H protein and in competition with the lipoylation; (3) both the lipoylation of H protein and its hydrolysis are enhanced by the apo and lipoylated forms of H protein and a mutant without the lipoylation site. A conceptual mechanistic model is proposed to explain these experimental observations in which conformational change of LplA upon interaction with H protein and competitive nucleophilic attacks play key roles.
Biopharmaceutical protein production using transgenic plant cell bioreactor processes offers advantages over microbial and mammalian cell culture platforms due to the ability to produce complex biologics, use of simple chemically-defined, animal component-free media, robustness of host cells, and biosafety. A disadvantage of plant cells from a traditional batch bioprocessing perspective is their slow growth rate which has motivated us to develop semicontinuous and/or perfusion processes. Although the economic benefits of plant cell culture bioprocesses are often mentioned in the literature, to our knowledge no rigorous techno-economic models or analyses have been published. Here we present techno-economic models in SuperPro Designer® for the large-scale production of recombinant butyrylcholinesterase (BChE), a prophylactic/therapeutic bioscavenger against organophosphate nerve agent poisoning, in inducible transgenic rice cell suspension cultures. The base facility designed to produce 25 kg BChE per year utilizing two-stage semicontinuous bioreactor operation manufactures a single 400 mg dose of BChE for $263. Semicontinuous operation scenarios result in 4-11% reduction over traditional two-stage batch operation scenarios. In addition to providing a simulation tool that will be useful to the plant-made pharmaceutical community, the model also provides a computational framework that can be used for other semicontinuous or batch bioreactor-based processes.
Acidithiobacillus ferrooxidans cells can oxidize iron and sulfur and are key members of the microbial biomining communities that are exploited in the large-scale bioleaching of metal sulfide ores. Some minerals are recalcitrant to bioleaching due to presence of other inhibitory materials in the ore bodies. Additives are intentionally included in processed metals to reduce environmental and microbially influenced corrosion. We have previously reported a new aerobic corrosion mechanism where A. ferrooxidans cells combined with pyrite and chloride can oxidize low grade stainless steel (SS304) with a thiosulfate-mediated mechanism. Here we explore process conditions and genetic engineering of the cells to enable corrosion of a higher grade steel (SS316). The addition of elemental sulfur and an increase in the cell loading resulted in a 74% increase in the corrosion of SS316 as compared to sulfur- and cell-free control experiments. The overexpression of the endogenous rus gene, which is involved in the cellular iron oxidation pathway, led to further 85% increase in the corrosion of the steel. Thus, the modification of the culturing conditions and cell line, led to a more than 3-fold increase in the corrosion of SS316 stainless steel, such that 15% of the metal coupons was dissolved in just 2 weeks. This work demonstrates how the engineering of cells and the optimization of their cultivation conditions can be used to discover conditions that lead to the corrosion of a complex metal target.
Biofilms commonly develop in flowing aqueous environments, where the flow causes the biofilm to deform. Because biofilm deformation affects the flow regime, and because biofilms behave as complex heterogeneous viscoelastic materials, few models are able to predict biofilm deformation. In this study, a phase field continuum model coupled with the Oldroyd-B constitutive equation was developed and used to simulate biofilm deformation. The accuracy of the model was evaluated using two types of biofilms: a synthetic biofilm, made from alginate mixed with bacterial cells, and a Pseudomonas aeruginosa biofilm. Shear rheometry was used to experimentally determine the mechanical parameters for each biofilm, as inputs for the model. Biofilm deformation under fluid flow was monitored experimentally using optical coherence tomography. The fit between the experimental and modeling geometries after fluid-driven deformation was very good, with relative errors of 12.8% for synthetic biofilm and 22.2% for homogenized P. aeruginosa biofilm. This is the first demonstration of the effectiveness of a viscoelastic phase field biofilm model. This model provides an important tool for predicting biofilm viscoelastic deformation. It also can benefit the design and control of biofilms in engineering systems.
Abstract This study describes the response of Arthrospira platensis to a variety of temperature conditions as reflected in variations of photosynthetic parameters, pigmentation, and biomass productivity in indoor photobioreactor (PBR) cultivations. These experiments are designed to better understand the impact of temperature, seasonal variations, and acclimation effects on outdoor biomass production. The irradiance level and temperature range (20 – 39°C) are chosen to enable modeling of semi-continuous operation of large-scale outdoor PBR deployments. Overall, the cultivations were quite stable with some pigment-related instabilities after prolonged high temperature exposure. Changes in productivity with temperature, as reflected in measured photosynthetic parameters, are immediate and mainly attributable to the temperature dependence of the photosaturation parameter, a secondary factor being variation in pigment content on a longer time scale corresponding to turnover of the culture population. Though pigment changes have minimum impact on productivity, prolonged exposure at 35°C and above yields a clear degradation in performance. Productivities in a semi-continuous operation are quantitatively reproduced with a productivity model incorporating photosynthetic parameters measured herein. This study confirms the importance of temperature for biomass and pigment production in Arthrospira cultivations and provides a basis for risk assessments related to temperature mitigation for large-scale outdoor cultivations. Keywords: Arthrospira Platensis, photosynthetic parameters, pigment production, productivity modeling, photobioreactors
Vaccines provide effective protection against many infectious diseases as well as therapeutics for some serious diseases, such as cancer. Many viral vaccines require amplification of virus in cell cultures during manufacture. Traditionally, cell cultures, such as VERO, have been used for virus production in bovine serum-containing culture media. However, due to concerns of potential adventitious agents present in fetal bovine serum (FBS), regulatory agencies suggest avoiding the use of bovine serum in vaccine production. Current serum-free media suitable for VERO-based virus production contains high concentrations of undefined plant hydrolysates. Although these media have been extensively used, the lack of chemical definition has potential to adversely affect cell growth kinetics and subsequent virus production. As plant hydrolysates are made from plant raw materials, performance variations could be significant among different lots of production. We developed a chemically defined, serum-free medium, OptiVERO, that was optimized specifically for VERO cells. VERO cell growth kinetics were demonstrated to be equivalent to EMEM-10% FBS in this chemically defined medium while the plant hydrolysate-containing medium demonstrated a higher doubling time in both 2D and 3D cultures. Virus production comparisons demonstrated that the chemically defined OptiVERO medium performed at least as good as the EMEM-10%FBS and better than the plant hydrolysate-containing media. We report the success in using recombinant proteins to replace undefined plant hydrolysates to formulate a chemically defined medium that can efficiently support VERO cell expansion and virus production.
Previously, our lab developed high molecular weight (MW) tense (T) state glutaraldehyde polymerized bovine hemoglobins (PolybHbs) that exhibited reduced vasoactivity in several small animal models. In this work, we prepared PolybHb in the T- and relaxed (R) quaternary state with ultrahigh MW (> 500 kDa) with varying cross-link densities and investigated the effect of MW on key biophysical properties (i.e., O2 affinity, cooperativity coefficient, hydrodynamic diameter, polydispersity, polymer composition, viscosity, gaseous ligand-binding kinetics, autoxidation, and haptoglobin-binding kinetics). To further optimize current PolybHb synthesis and purification protocols, we performed a comprehensive meta-data analysis to evaluate correlations between procedural parameters (i.e. cross-linker:bovine Hb (bHb) molar ratio, gas/liquid exchange time, temperature during dithionite addition, and number of diafiltration cycles) and the biophysical properties of both T-state and R-state PolybHbs. Our results showed that, the duration of the fast-step autoxidation phase of R-state PolybHb increased with decreasing glutaraldehyde:bHb molar ratio. Additionally, T-state PolybHb exhibited significantly higher biomolecular rate constants for binding to haptoglobin and unimoleular O2 offloading rate constants compared to R-state PolybHb. The methemoglobin (metHb) level in the final product was insensitive to the molar ratio of glutaraldehyde to bHb for all PolybHb. During tangential flow filtration processing of the final product, 14 diafiltration cycles was found to yield the lowest metHb level.
Inflammatory breast cancer (IBC), a rare form of breast cancer associated with increased angiogenesis and metastasis, is largely driven by tumor-stromal interactions with the vasculature and the extracellular matrix (ECM). However, there is currently a lack of understanding of the role these interactions play in initiation and progression of the disease. In this study, we developed the first three-dimensional, in vitro, vascularized, IBC platform to quantify the spatial and temporal dynamics of tumor-vasculature and tumor-ECM interactions specific to IBC. Platforms consisting of collagen type 1 ECM with an endothelialized blood vessel were cultured with IBC cells, MDA-IBC3 (HER2+) or SUM149 (triple negative), and for comparison to non-IBC cells, MDA-MB-231 (triple negative). An acellular collagen platform with an endothelial blood vessel served as control. SUM149 and MDA-MB-231 platforms exhibited a significantly (p<0.05) higher vessel permeability and decreased endothelial coverage of the vessel lumen compared to the control. Both IBC platforms, MDA-IBC3 and SUM149, expressed higher levels of VEGF (p<0.05) and increased collagen ECM porosity compared to non-IBC MDA-MB-231 (p<0.05) and control (p<0.01) platforms. Additionally, unique to the MDA-IBC3 platform, we observed progressive sprouting of the endothelium over time resulting in viable vessels with lumen. The newly sprouted vessels encircled clusters of MDA-IBC3 cells replicating a feature of in vivo IBC. The IBC in vitro vascularized platforms introduced in this study model well-described in vivo and clinical IBC phenotypes and provide an adaptable, high throughout tool for systematically and quantitatively investigating tumor-stromal mechanisms and dynamics of tumor progression.
Recently, numerous synthetic small molecular peptidomimetics have been designed to overcome the shortcomings of antimicrobial peptides (AMPs), such as protease instability and high production cost. TZP4 is a triazine-based amphipathic polymer designed to mimic the amphipathic structure found in AMPs. Compared to melittin, TZP4 showed superior antimicrobial activity against antibiotic-resistant pathogens, such as methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa. Results of membrane depolarization, SYTOX Green uptake, flow cytometry, and gel retardation assays suggested that the mechanism of antimicrobial action of TZP4 involved an intracellular target rather than the bacterial cell membrane. Furthermore, TZP4 suppressed the mRNA levels of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-α (TNF-α) and inhibited the release of NO and TNF-α in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. BODIPY-TR-cadaverine displacement and dissociation of Fluorescein isothiocyanate (FITC) labelled lipopolysaccharides (LPS) assays revealed that TZP4 strongly bound to Escherichia coli-derived LPS and disaggregated the LPS oligomers. Additionally, flow cytometric analysis revealed that TZP4 inhibited the binding of FITC-conjugated LPS to RAW264.7 cells. These observations indicate that TZP4 may exert its anti-endotoxin activity by directly binding with LPS and inhibiting the interaction between LPS and CD14+ cells. Thus, we propose that TZP4 is a promising drug candidate for the treatment of endotoxic shock and sepsis caused by gram-negative bacterial infections.
Dibutyl phthalate (DBP) is an environmental pollutant that can threaten human health. The strain Arthrobacter sp. ZJUTW, isolated from the sludge of river of Hangzhou city, can efficiently degrade DBP. Its genomic and transcriptomic differences when cultivated with DBP and with glucose revealed specific DBP metabolic pathways in the ZJUTW strain. The degrading gene clusters distribute separately on a circular chromosome and a plasmid pQL1. Genes related to the initial steps of DBP degradation from DBP to phthalic acid (PA), the pehA gene and pht gene cluster, are located on the plasmid pQL1. While pca gene cluster related to the transforming of protocatechuic acid (PCA) to acetyl-CoA, is located on the chromosome. After homologous alignment analysis with the reported gene clusters, we found that there were a series of double copies of homologous genes in pht and pca gene clusters that contribute to the efficient degradation of DBP by ZJUTW. In addition, transcriptomic analysis showed a synergistic effect between pht and pca clusters, which also favor ZJUTW allowing it to efficiently degrade DBP. Combined genomic and transcriptomic analyses affords the complete DBP metabolic pathway in Arthrobacter sp. ZJUTW that is different from that of reported other Arthrobacter strains. After necessary modification based on its metabolic characteristics, Arthrobacter sp. ZJUTW or its mutants might represent promising candidates for use in the bioremediation of DBP pollution.
Glutathione (GSH) plays a central role in the redox balance maintenance in mammalian cells. The study of industrial CHO cell lines have demonstrated a close link between GSH metabolism and clone productivity. However, a deep investigation is still required to understand this correlation and highlights new potential targets for cell engineering. In this study, we have modulated the GSH intracellular content of an industrial cell line under bioprocess conditions in order to further elucidate the role of the GSH synthesis pathway. Two strategies were used : the variation of cystine supply and the direct inhibition of the GSH synthesis using buthionine sulfoximine (BSO). Cysteine supply modulation have revealed a correlation between intracellular GSH and product titer over time. Analysis of metabolites uptake/secretion rates and proteome comparison between BSO-treated cells and non-treated cells has highlighted a slow down of the TCA cycle leading to a secretion of lactate and alanine in the extracellular environment. Moreover, an adaptation of the glutathione related proteome has been observed with a up-regulation of the regulatory subunit of glutamate cysteine ligase and a down-regulation of a specific glutathione transferase subgroup, the Mu family. Surprisingly, the main impact of BSO treatment was observed on a global down-regulation of the cholesterol synthesis pathways. As cholesterol is required for protein secretion, it can be the missing part of the jigsaw to finally elucidate the link between GSH synthesis and productivity.
In order to maximize the productivity of engineered metabolic pathway, in silico model is an established means to provide features of enzyme reaction dynamics. In our previous study, E.coli engineered with acrylate pathway yielded low propionic acid titre. To understand the bottleneck behind this low productivity, a kinetic model was developed that incorporates the enzymatic reactions of the acrylate pathway. The resulting model was capable of simulating the fluxes reported under in vitro studies with good agreement, suggesting repression of propionyl-CoA transferase by carboxylate metabolites as the main limiting factor for propionate production. Furthermore, the predicted ﬂux control coeﬃcients of the pathway enzymes under steady state conditions revealed that the control of ﬂux is shared between propionyl-CoA transferase and lactoyl-CoA dehydratase. Increase in lactate concentration showed gradual decrease in ﬂux control coeﬃcients of propionyl-CoA transferase that in turn confirmed the control exerted by the carboxylate substrate. To interpret these in silico predictions under in vivo system, an organized study was conducted with a Lactic Acid Bacteria (LAB) strain engineered with acrylate pathway. Analysis reported a decreased product formation rate on attainment of inhibitory titre by suspected metabolites and supported the model.
Fluorescent in situ hybridization (FISH) has been extensively used in the past decades for the detection and localization of nucleic acid sequences or of the microorganisms themselves within samples. However, a mechanistic approach of the whole FISH process is still missing, and the main limiting steps for the hybridization to occur remain unclear. In here, FISH is approached as a particular case of a diffusion-reaction kinetics, where molecular probes move from the hybridization solution to the target RNA site within the cells. Based on literature models, the characteristic times taken by different molecular probes to diffuse across multiple cellular barriers, and the reaction time associated with the formation of the duplex molecular probe-RNA were estimated. Structural and size differences at the membrane level of bacterial and animal cells were considered. For bacterial cells, the limiting step for diffusion is likely to be the peptidoglycan layer (characteristic time of 2700-4524 s), whereas for animal cells the limiting step should be the diffusion of the probe through the bulk (1.8-5.0 s) followed by the diffusion through the lipid membrane (1 s). The information provided here may serve as a basis to optimize FISH protocols.
Shewanella oneidensis MR-1, a model strain of exoelectrogenic bacteria (EEB), plays a key role in environmental bioremediation and bioelectrochemical systems because of its unique respiration capacity. However, only a narrow range of substrates can be utilized by S. oneidensis MR-1 as carbon sources, resulting in its limited applications. In this work, a rapid, highly efficient and easily manipulated base editing system pCBEso was developed by fusing a Cas9 nickase (Cas9n (D10A)) with the cytidine deaminase rAPOBEC1 in S. oneidensis MR-1. The C-to-T conversion of suitable C within the base editing window could be readily and efficiently achieved by the pCBEso system without requiring double strand break or repair templates. Moreover, double-locus simultaneous editing was successfully accomplished with an efficiency of 87.5. With this tool, the roles of the key genes involving in N-acetyl-glucosamine (GlcNAc) or glucose metabolism in S. oneidensis MR-1 were identified. Furthermore, an engineered strain with expanded carbon source utilization spectra was constructed and exhibited a higher degradation rate for multiple organic pollutants (i.e., azo dyes and organoarsenic compounds) than the wild type when glucose or GlcNAc was used as the sole carbon source. Such a base editing system could be readily applied to other EEB. This work not only enhances the substrate utilization and pollutant degradation capacities of S. oneidensis MR-1, but also accelerates the robust construction of engineered strains for environmental bioremediation.
Tissue constructs of physiologically relevant scale require a vascular system to maintain cell viability. However, in vitro vascularization of engineered tissues is still a major challenge. Successful approaches are based on a feeder layer (FL) to support vascularization. Here, we investigated whether the supporting effect on the self-assembled formation of vascular-like structures by microvascular endothelial cells (mvECs) originates from the FL itself or from its extracellular matrix (ECM). Therefore, we compared the influence of ECM, either derived from adipose-derived stem cells (ASCs) or adipogenic differentiated ASCs, with the classical approaches based on a cellular FL. All cell-derived ECM (cdECM) substrates enable mvEC growth with high viability. Vascular-like structure formation was visualized by immunofluorescence staining of endothelial surface protein CD 31 and can be observed on all cdECM and FL substrates but not on control substrate collagen I. On adipogenic differentiated ECM longer and higher branched structures can be found compared to stem cell cdECM. An increased concentration of pro-angiogenic factors can be found in cdECM substrates and FL approaches compared to controls. Finally, expression of proteins associated with tube formation (E-selectin and thrombomodulin) was confirmed. These results highlight cdECM as promising biomaterial for in vitro vascularization in adipose tissue engineering.